You are on page 1of 46

1

Pharmacological Evaluation of Different lettuce Varities


for their medicinal Properties

By

Hina Hajra Munir

Department of biochemistry

Faculty of biological sciences


2
MPhil 2015-2017
Introduction

• Lactuca sativa an edible crop belongs to Asteraceae family


• An important vegetable in group of leafy vegetables
• Consumed as a raw form (El-Said, 2012)
• Contains moisture and is low in calories
• Calcium, iron, sodium, copper, potassium, vitamin K, A, E and
phosphorous (Koopman et al., 1998)
• Self pollination with the annual growth (Ryder, 1979)
• Hair less leaves (Vries,1997)

3
Selected Lettuce varieties for biological evaluation

Variety name Aqueous extract Ethyl acetate extract


Hearts of romaine A2 E2
Sweet gem lettuce A3 E3
Iceberg lettuce A4 E4
Little gem lettuce A6 E6
Hearts of cos A7 E7
Cos lettuce A8 E8
British curly leaf lettuce A9 E9
Irish lettuce A10 E10
lamb’s lettuce A11 E11
British Chinese leaf lettuce A12 -
Red mini cos lettuce A13 E13
4
Cont…

Fig. 1.1: Represents different varieties of lettuce (a) Hearts of romaine


(b) Sweet gem lettuce (c) Iceberg lettuce (d) little gem lettuce (e)Hearts of
cos (f) Cos lettuce (g) British curly leaf lettuce (h) Irish lettuce (i) Lambs
lettuce (j) British Chinese leaf lettuce (k) Red mini cos lettuce.
5
Bioassay

• Preliminary screening procedure


• Evaluation of biological activity
• Compares with standard
• Plays an important role in drug discovery and development

6
Research Objectives

To determine the pharmacological activity of different


lettuce varieties against following bioassays
• Enzyme inhibition activities
• Antimicrobial activities
• Cytotoxic potential
• Antitumor activities
• DNA binding abilities

7
Enzyme Inhibition Assays

• α-glucosidase inhibition assay


• α-amylase inhibition assay
• Butrylcholinesterase inhibition assay
• Acetylcholinesterase inhibition assay

8
α-glucosidase inhibition assay

• Digest carbohydrates
• Facilitates absorption of glucose from intestine

α-glucosidase

starch and disaccharides

• Inhibitors of enzyme antagonizes its activity


• and decreases the blood glucose level
• role in diabetes

9
(Matsui et al., 2007)
Cont…
Sr. No. Sample Percentage inhibition IC50 µg/ml
1000 µg/ml 500 µg/ml 250 µg/ml
1 A2 55.0±2.6 20.2±2.8 4.0±0.6 914.9
2 E2 34.4±4.0 13.3±2.8 2.7±0.6 1405
3 A3 38.3±1.1 15.1±1.3 2.2±1.1 1268
4 E3 71.3±4.1 29.7±2.6 5.9±0.6 701.6
5 A4 12.7±1.5 5.5±0.6 1.2±0.1 3654
6 E4 64.2±1.8 26.9±0.5 5.5±0.1 776.0
7 A6 18.3±1.7 6.6±1.4 1.0±0.7 2288
8 E6 66.7±3.9 23.1±5.9 4.9±1.6 775.3
9 A7 31.1±4.8 15.8±1.9 3.0±0.6 1658
10 E7 58.4±4.6 25.4±1.9 5.2±0.3 847.4
11 A8 27.5±1.3 12.3±1.2 2.4±0.1 1805
12 E8 28.4±4.7 11.4±2.1 2.3±0.4 1687
13 A9 26.0±3.1 10.9±1.3 2.3±0.2 1865
14 E9 74.8±1.7 32.4±1.2 6.4±0.1 662.4
15 A10 60.3±0.6 23.4±2.7 4.3±0.9 835.4
16 E10 72.8±1.5 30.7±0.6 6.3±0.1 685.0
17 A11 41.3±4.3 17.2±2.3 3.7±0.5 1200
18 E11 33.2±2.0 13.5±1.7 2.9±0.1 1472
19 A12 51.8±3.8 22.3±1.5 4.4±0.4 957.5
20 A13 25.8±4.8 9.8±2.8 2.1±0.4 1806
21 E13 49.5±2.5 21.6±0.9 4.4±0.1 1002

10
α-amylase inhibition assay

• starch α-amylase maltose


• dark blue presence of starch
• yellow colour absence of starch
• brownish colour partially degraded starch

+ive controls

-ive control
11
(Keerthana et al., 2013)
Cont…
Sr. No. Sample Percentage inhibition IC50 µg/ml
1000 µg/ml 500 µg/ml 250 µg/ml
1 A2 15.2±2.2 5.9±1.0 2.3±0.3 3161
2 E2 25.7±4.0 12.3±1.9 6.0±0.8 2337
3 A3 12.1±2.0 5.8±0.8 2.3±1.2 5091
4 E3 28.4±2.2 13.7±1.0 6.4±5.0 2050
5 A4 10.4±0.4 4.8±0.1 2.1±2.4 5923
6 E4 24.3±0.3 11.3±0.1 5.4±4.3 2427
7 A6 9.8±0.9 4.3±0.2 2.2±2.2 6354
8 E6 31.2±2.1 17.1±1.2 8.3±0.9 1982
9 A7 7.9±1.7 2.4±0.9 1.1±1.2 4297
10 E7 35.5±1.9 16.4±0.7 9.1±3.1 1585
11 A8 10.7±2.2 5.1±1.1 2.5±0.2 6517
12 E8 23.0±2.1 10.1±1.5 4.8±1.0 2464
13 A9 9.0±1.0 3.8±0.6 1.9±2.1 6377
14 E9 30.5±0.7 14.7±0.2 7.2±4.2 1900
15 A10 9.3±0.2 3.9±0.1 1.8±1.1 5838
16 E10 29.0±2.7 16.7±1.3 6.6±3.2 2124
17 A11 9.6±0.3 4.0±0.1 1.5±0.4 5126
18 E11 38.0±2.6 16.9±1.4 7.5±1.4 1386
19 A12 8.0±0.6 3.5±0.2 1.3±0.2 6485
20 A13 8.4±0.4 4.1±0.1 2.1±0.7 9315
21 E13 32.1±5.1 16.3±2.9 8.3±3.1 1796
12
Butrylcholinesterase inhibition assay

butrylthiocholine Butrylcholinesterase thiocholines DNTB

5-thio-2-nitrobenzoate anion (yellow colour)

-ive control

13
(Jabeen et al., 2014)
Butrylcholinesterase assay

Sr. No. Sample Percentage inhibition IC50 µg/ml


5mg/ml 2.5mg/ml 1.25mg/ml
1 A2 44.4±2.3 21.3±1.0 8.0±0.1 1151
2 E2 84.0±1.7 41.7±1.1 20.4±0.0 531.9
3 A3 99.1±0.8 49.8±3.2 23.6±0.3 448.8
4 E3 43.8±0.2 19.8±2.2 7.7±1.1 1167
5 A4 65.6±1.9 29.9±0.0 11.5±3.2 737.8
6 E4 45.6±1.9 22.1±3.2 9.8±1.4 1129
7 A6 57.7±3.1 24.4±3.3 10.5±1.3 862.2
8 E6 62.7±0.6 30.2±1.2 11.6±4.1 765.2
9 A7 54.7±2.0 23.6±2.1 7.3±0.4 908.6
10 E7 98.7±1.2 45.6±2.1 20.5±0.2 711.4
11 A8 60.3±4.6 25.4±3.2 10.6±2.1 1475
12 E8 97.4±0.9 46.3±2.1 22.3±3.2 806.3
13 A9 59.3±0.0 23.4±0.1 9.0±1.0 1489
14 E9 84.3±4.2 39.8±1.0 9.7±0.2 850.7
15 A10 51.9±4.2 22.6±1.9 7.5±2.1 1691
16 E10 55.5±4.8 20.9±2.0 6.6±2.0 1205
17 A11 52.3±4.7 11.6±2.0 5.0±0.5 1427
18 E11 98.5±1.3 44.7±1.4 19.6±0.5 705.2
19 A12 50.9±3.0 18.5±3.7 5.0±1.7 1630
14
20 A13 71.0±1.9 33.6±2.0 9.6±3.0 1291
Acetylcholinesterase assay

Acetylthiocholine Acetylcholinesterase thiocholines DNTB

5-thio-2-nitrobenzoate anion (yellow colour)

-ive control

15
Cont…
Sr. No. Sample Percentage inhibition IC50 µg/ml
5mg/ml 2.5mg/ml 1.25mg/ml
1 A2 93.4±0.0 44.2±1.2 19.2±0.2 500.7
2 E2 82.0±3.0 39.1±3.9 11.7±0.2 578.6
3 A3 62.4±3.3 25.6±3.1 10.7±0.3 796.8
4 E3 100±0.0 47.2±1.1 18.7±2.1 480.4
5 A4 91.3±2.2 40.3±2.1 15.4±2.0 537.5
6 E4 90.3±2.9 35.8±1.1 9.9±0.3 574.2
7 A6 93.1±3.3 41.3±2.1 20.1±1.1 514.3
8 E6 100±0.0 49.4±1.1 19.9±1.1 464.7
9 A7 77.2±0.8 33.2±1.4 12.2±0.4 634.4
10 E7 100±0.0 47.1±1.4 22.3±0.3 466.7
11 A8 63.4±2.5 32.3±1.2 10.3±0.4 746.1
12 E8 100±0.0 50.1±2.1 28.6±0.2 424.9
13 A9 100±0.0 45.4±1.1 14.2±1.0 504.5
14 E9 100±0.0 48.6±1.1 23.3±1.1 455.0
15 A10 100±0.0 47.2±4.3 20.4±1.9 473.9
16 E10 70.7±4.6 32.4±2.1 14.4±2.2 674.3
17 A11 70.6±3.9 31.3±2.1 15.3±1.1 680.0
18 E11 100±0.0 49.0±2.1 20.1±1.9 465.9
19 A12 52.8±0.7 18.3±1.2 4.2±2.1 952.7
20 A13 98.2±1.8 39.9±2.7 15.8±2.1 531.7
21 E13 100±0.0 50.4±2.1 18.2±1.0 466.0 16
Antimicrobial Assays

• Infection in animals and humans by microorganisms


• Treatment of infection by antimicrobials
• Development of resistance to antimicrobials
• Search for new appropriate antimicrobial agents
• Determination of inhibitory activity of different lettuce
varieties against microorganisms
• Disc diffusion method applies on
Antibacterial assay
Antifungal assay

17
Antibacterial assay

• Assay was performed on four bacterial strains


Two were gram positive
• Micrococcus luteus
• Staphylococcus aureus

Two were gram negative


• Enterobacter aerogens
• Escherichia coli

18
(Bauer et al., 1966)
Cont…

Sr. No. Sample. Code Enterobacter aerogens Escherichia coli Micrococcus luteus
Staphylococcus aureus

Zone of inhibition at highest concentration

(mm)
1 A2 . - . 9.1±1.1
2 E2 . . . 18.5±0.3
3 A3 . . . 16.0±0.7
4 E3 . . . 8.3±0.9
5 A4 . . . 8.5±1.0
6 E4 . . . 8.3±1.1
7 A6 . . . 5.2±1.2
8 E6 9.6±0.9 . . 6.8±0.9
9 A7 . 17.6±0.6 . .
10 E7 . . . 6.9±1.0
11 A8 . . . .
12 E8 . . . .
13 A9 . . . .
14 E9 . . . .
15 A10 . . .
16 E10 . . . 8.7±0.7
17 A11 . . . .
18 E11 . . . .
19 A12 . . . .
20 A13 . . . .
21 E13 . . 11.6±1.1 . 19
22 Kanamycin 29.2 26.1 29.1 29.1
Micrococcus luteus

20
Escherichia coli

21
Staphylococcus aureus

22
Enterobacter aerogens

23
Antifungal assay

Disc diffusion assay


Against five fungal strains

• Mucor species
• Fusarium solani
• Aspergillus flavus
• Aspergillus fumigates
• Aspergillus niger

24
(Esteban et al ., 2005)
Cont…
Aspergillus fumigates no activity
Aspergillus niger no activity
Fusarium solani no activity
Mucor species no activity
Aspergillus flavus activity

S sample Aspergillus Aspergillus Aspegillus Fusarium Mucor


r
flavus fumigates niger solani species
Zone of inhibition at highest concentration (mm)
1 A10 4.4±0.2 - - - -
2 turbinafine 5.35 4.90 4.17 1.62 1.52

25
Aspergillus fumigates

26
Mucor species

27
Fusarium solani

28
Aspergillus niger

29
Aspergillus flavus

30
Brine Shrimp Cytotoxicity Assay

• Simple and inexpensive method


• Cytotoxic lethality assay
• Based on the ability of lettuce varieties to kill Nauplii of the
brine shrimp Artemia salina
• Illustrates cytotoxic, pesticidal and anticancer activities

31
(Mclaughlin and Rogers 1998)
Cont…

• No cytotoxicity

lettuce varieties No toxicity

32
Potato Disc Antitumor Assay

• This assay illustrates the antitumor potential of different


lettuce varieties
• Based on the ability of test sample to inhibit tumour formation
in potatoes
• Agrobacterium tumefaciens responsible for tumour formation.

33
(Ahmad et al ., 2008)
Cont…

Antibacterial assay as a pre-screening test

no activity

34
Cont…
Sr. No. Sample Percentage inhibition IC50 µg/ml

1000 µg/ml 100 µg/ml 10 µg/ml


1 A2 46.6±2.2 21.6±1.1 9.0±1.1 1093
2 E2 68.2±2.2 39.1±2.2 9.6±0.3 658.3
3 A3 46.6±2.2 21.6±1.1 9.0±1.1 1093
4 E3 70.5±4.3 34.6±2.2 13.2±2.1 664.3
5 A4 65.8±2.1 32.6±2.1 12.8±2.2 716.6
6 E4 64.2±1.8 26.9±0.5 5.5±0.1 776.0
7 A6 34.3±2.1 12.3±1.6 5.5±2.1 1453
8 E6 25.7±4.0 12.3±1.9 6.0±0.8 2337
9 A7 69.0±2.6 30.7±1.0 13.6±0.3 699.7
10 E7 17.3±0.2 2.8±0.0 1.0±0.0 1760
11 A8 38.0±0.6 14.3±2.0 6.6±1.0 1336
12 E8 29.0±2.7 16.7±1.3 6 .6±3.2 2124
13 A9 9.6±0.3 4.0±0.1 1.5±0.4 5126
14 E9 36.4±1.7 14.9±2.0 6.6±1.9 1421
15 A10 89.6±2.1 23.4±2.7 4.3±0.9 835.4
16 E10 8.7±2.1 4.1±1.1 1.0±0.1 5788
17 A11 31.4±0.5 9.7±0.5 1.8±0.3 1439
18 E11 90.3±2.9 35.8±1.1 9.9±0.3 574.2
19 A12 44.4±3.7 19.6±2.0 3.2±1.0 636.6
20 A13 21.4±0.4 9.6±1.0 3.0±1.0 2447
21 E13 39.1±0.1 30.4±0.0 18.9±0.0 2415

35
DNA-Drug Interaction Assay

• Interaction of ligands with DNA


• Ligand molecules can be used as DNA targeted drugs
• DNA as an effective target against antiviral, antibiotic and
anticancer drugs

36
(Arslantas et al., 2007)
Cont…

• spectrophotometer
• Addition of DNA in pure sample results in change in
absorbance or peak shift

• Red shift longer wavelength


bathochromic effect

• Blue shift shorter wavelength


hyperchromic effect

• Hyperchromism

37
Cont…

2
Absorbance

1.5

0.5

0
230 280 330 380

Wavelength (nm)

Absorption spectrum of DNA dissolved in double


distilled water

38
1
0.9 0.5 e-6M DNA + extract
0.8
Absorbance
0.7 1 e-6M DNA + extract
0.6
0.5 e-5M DNA + extract
0.5
0.4 1e-5M DNA + extract
0.3
only extract
0.2
0.1
0
250 270 290 310 330 350 370 390 410 430 450
Wavelength (nm)

Absorption spectrum of A2 in the absence and presence of


DNA; the absorbance changes upon increasing DNA
concentration

39
4

3.5

2.5
Absorbance

Only extract
2 0.5e-6M DNA + extract
1e-6M DNA + extract
1.5 0.5e-5M DNA + extract
1e-5M DNA + extract
1

0.5

0
320 370 420 470 520 570
Wavelength (nm)

Absorption spectrum of A6 in the absence


and presence of DNA; the absorbance and
peak shift changes upon changing the DNA
concentration
40
1.4

1.2

1
Absorbance

Only extract
0.8
0.5e-6M DNA + extract
1e-6M DNA + extract
0.6
0.5e-5M DNA + extract
1e-5M DNA + extract
0.4

0.2

0
260 310 360 410 460
Wavelength (nm)

Absorption spectrum of A10 in the absence and presence of


DNA; the absorbance increases upon increasing DNA
concentration
41
Conclusion

• These extracts exhibit variant degree of activity in all the


bioassays
• Aqueous extracts showed more activity then the ethylacetate
extracts
• Further research could be carried on different bioassays using
same varieties to explore new and effective drugs

42
Acknowledgment

• I feel highly privileged in taking opportunity to express my


profound gratitude to my supervisor professor Dr. Bushra
Mirza for her skilful guidance, motivational support and keen
interest in my work. Her guidance helped me in all the time of
research and writing of this thesis.
• Thankful to my phd senior Hammad Ismail for his co-
supervision
• All lab fellows and clerical staff

43
References
• Ahmad MS, Hussain M, Hanif M, Ali S, Qayyum M and Mirza B (2008). Di‐and
Triorganotin (IV) esters of 3, 4‐Methylenedioxyphenylpropenoic acid: synthesis,
spectroscopic characterization and biological screening for antimicrobial,
cytotoxic and antitumor activities. Chem Biol Drug des 71(6): 568-576.
• Arslantas A, Devrim AK and Necefoglu H (2007). The interaction of sheep
genomic DNA with a cobalt (II) complex containing p-nitrobenzoate and n,n`-
diethylnicotinamide ligands. Int J Mol Sci 8(12): 1225-1233.
• Bauer AW, Kirby WMM, Sherris JC and Turck M (1966). Antibiotic susceptibility
testing by a standardized single disk method. Am J Clin Pathol 45(4): 493-496.
• El-Said, SD (2012). Detection of parasites in commonly consumed raw
vegetables. Alexandria Med J 48(4): 345-352.
• Esteban A, Abarca ML and Cabanes FJ (2005). Comparison of disk diffusion
method and broth microdillution method for antifungal susceptibility testing of
dermatophytes. Med Mycol 43(1): 61-66.

44
Cont…

• Jabeen F, Oliferenko PV, Oliferenko AA, Pillai GG, Ansari FL, Hall CD and
Katritzky AR (2014). Dual inhibition of the α-glucosidase and butrylcholinesterase
studied by Molecular Field Topology Analysis. Eur J Med Chem 80: 228-242.
• Keerthana G, Kalaivani MK and Sumathy A (2013). In-vitro alpha amylase
inhibitory and anti-oxidant activities of ethanolic leaf extract of Croton
bonplandianum. Asian J Pharm Clin Res 6(4): 32-36.
• Koopman WJ, Guetta E, van de Wiel CC, Vosman B and van den Berg RG (1998).
Phylogenetic relationships among Lactuca (Asteraceae) species and related genera
based on ITS-1 DNA sequences. Am J Bot 85(11): 1517-1530.
• Matsui T, Tanaka T, Tamura S, Toshima A, Tamaya K, Miyata Y, Tanaka K and
Matsumoto K (2007). α-Glucosidase inhibitory profile of catechins and theaflavins.
J Agric Food Chem 55: 99-105.
• McLaughlin JL and Rogers LL (1998). The use of biological assays to evaluate
botanicals. Drug Inform J 32: 513-524.
• Ryder E (1979). Leafy salad vegetables. A VI Publishing Co, Westport, CT.
• Vries IM (1997). Origin and domestication of Lactuca sativa L. Genet Resour and
crop Ev 44(2): 165–164.
45
46

You might also like